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. 2021 Oct 8;12:5913. doi: 10.1038/s41467-021-25944-2

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Skin sections of 7-week old OTULIN^fl/fl and Δ^KerOTULIN mice stained with antibodies against cleaved caspase-3 and Ki-67 to assess apoptosis and proliferation, respectively. NL non lesional; L lesional. Scale bar: 200 μm. b Quantification of the number of interfollicular epidermis (IFE) cells from 7-week old OTULIN^fl/fl (WT) and Δ^KerOTULIN (KO) mice that stain positive for cleaved caspase-3 and Ki-67 (n = 9 WT mice; n = 6 and 4 KO non-lesional mice per condition; n = 6 and 4 KO lesional mice; One-way ANOVA; *p < 0.5; **p < 0.01; ****p < 0.0001). Data represent means ± SEM. c Western blot analysis for expression of full-length (FL) and cleaved (Cl) caspase-3 in epidermal tail lysates from OTULIN^fl/fl and Δ^KerOTULIN mice. Anti-actin immunoblotting was used as a loading control. Molecular weight marker units are in kilodalton (kD). This experiment was repeated three times independently with similar results. d EdU (green) retaining cells in tail wholemount sections of 7-week old OTULIN^fl/fl and Δ^KerOTULIN mice after 3 h chase. Wholemounts were stained for Keratin-14 (K14; red) and counterstained with Dapi (blue). Scale bars: 100 μm. Lower panels show EdU staining only. This experiment was repeated three times independently with similar results. e Tail wholemount sections of 7-week old OTULIN^fl/fl and Δ^KerOTULIN mice stained for cleaved caspase-3 (red) and counterstained with Dapi. Lower panels depict magnified views. Eight mice per genotype were analyzed. Scale bars: 50 μm. f, g Quantification of epidermal thickness (f) and the number of cleaved caspase-3-positive cells (g) in the IFE of skin sections of P0.5 control and Δ^KerOTULIN pups (WT: n = 3; KO: n = 4; Mann−Whitney two-sided test; ***p = 0.0007). Data represent means ± SEM. h Representative images of cleaved capase-3-stained skin sections of OTULIN^fl/fl and Δ^KerOTULIN pups. Eight mice per genotype were analyzed. Arrows indicate apoptotic IFE cells. Lower panels depict magnified views of the boxed areas. Scale bars: 100 μm.